【作者】 吕学东；

【导师】 江汉湖；

【作者基本信息】 南京农业大学 ， 食品工程， 2004， 硕士

【摘要】 对硫磷残留是大米安全卫生的重要检测项目之一，鉴于目前大米中对硫磷残留的测定方法(主要有气相色谱法等)不便于基层单位对大米中对硫磷残留进行大批次、低成本的快速、有效监测，本研究参考国外先进技术，依据抗原与抗体可发生特异性反应的特点，人工制备了抗对硫磷抗体，运用便捷、灵敏、准确的ELISA技术定量检测大米中的对硫磷残留，取得满意结果。摘要如下： 1、本研究采用重氮化法，将对硫磷分别与BSA和OVA偶联，经提纯并以紫外光谱扫描等鉴定，成功地制备了2种对硫磷完全抗原，其中对硫磷-BSA结合物作为免疫原，对硫磷-OVA结合物作为包被抗原。将免疫原接种新西兰兔，适时提取抗血清，以对硫磷-OVA结合物包被微量反应条，采用间接ELISA测试，发现采用对硫磷-BSA结合物免疫所获的抗血清效价＞1：409600。以此抗血清作为试验用抗体。 2、通过条件试验，确定了所获抗血清和包被抗原的最佳工作浓度分别为1：6000和1：25600。以此工作浓度，采用间接ELIsA抑制试验测试，显示当对硫磷标液浓度由39ng／ml倍比递增至5000ng／ml时，抑制反应呈现较好的线性关系，其标准抑制曲线的相关系数为0.993，表明所获抗血清可与对硫磷发生特异性结合。从而初步确定了以间接ELIsA抑制试验来定量检测对硫磷的方法。 3、为考证方法的可靠性，还采用如下试验进行了验证： (1)特异性试验：用本方法测试其他有关化合物的交叉反应，显示对硫磷抗血清与还原对硫磷有强反应，但该化合物是合成对硫磷—BSA时的中间产物，自然界并不存在。与甲基对硫磷和对氧磷有轻度交叉，但在浓度分别为0.1 mg／ml和0.2 mg／ml时交叉反应仅2.5％。与对硝基酚、马拉硫磷、杀螟硫磷、乐果无交叉反应。 (2)准确度试验：将添加有0ng／g-5000ng／g系列浓度对硫磷标准品的大米样品共5份经简单处理，以本法测试，4小时可获结果，其回收率为81-103％，平均为89.3％。将上述样品共5份，委托外单位以高效气相色谱法检测，显示本法所获结果与色谱法结果的符合率为76-100％，平均为89.8％。 (3)精密度试验：将添加有0ng／g-5.0ug／g系列浓度对硫磷标准品的大米样品共5份进行7次测试，经数理统计分析，其结果的平均批间误差为6.1％。再分别在一大米中对硫磷残留量免疫学快速检测方法的研究个实验室由3名操作人员进行测试，其结果的室内变异系数平均为5.2%. 试验显示，本方法简便、快速、灵敏、准确，适用于大米中对硫磷残留的检测。查新表明，本方法属国内首创。更多还原

【Abstract】 Parathion residue is one of the important detecting items of safety in rice. However, routine detection methods for parathion residues of rice (including HPGC etc.) is not fit for rapid & available inspection of parathion residues of numbers of rice. Therefore, we, consulting advanced technology overseas on the study, according as peculiarity of specific reaction between antigen and antibody, preparing anti-parathion antibody, detecting parathion residues of rice with imply, sensitivity and precise ELISA technology, and obtained satisfactory results as follows:1 , Parathion were coupled to carried protein BSA or OVA by means of the "-N=N-" respectively. By purification and identify with scanning of UV-VIS spectra, two kinds of complete antigen were preparing. Therein, parathion-BSA conjugate are acted as immunogen, and parathion-OVA conjugate as coated antigen. The antiserum was collected from the New Zealand rabbits immunized with above immunogen at an appropriate time, and measured with indirect ELISA on microtiter wells coated with parathion-OVA conjugate. The liters of antiserum from one the rabbits immunized with parathion -BSA conjugate are over 1:409600. This antiserum is used as antibody on study.2, The best concentration of the antiserum and the coated antigen were fixed on 1:6000 and 1:25600 by tests on condition, respectively. With inhibitory assay of indirect ELISA in the concentration, the inhibitory reaction is the better liner with the series of the concentration of parathion standard solutions from 39ng/ml to 5000ng/ml in double time. The correlative coefficient of standard inhibitory curve is 0.993,which show specific reaction between antiserum and parathion. Thereby, with inhibitory assay of indirect ELISA, the quantitative methods detecting parathion was set up.3 >. In order to validating the reliability of the methods, following tests were made.(1) Specificity test: Measuring other drugs with the methods, the antiserum cross-reactivity with reducing parathion, methyl-parathion and paraoxon are 100%, 2.5% and 2.5%, respectively. But the antiserum could not react with p-nitrophenol, malathion, fenitrothion and dimethoate.(2) Accuracy test: 5 samples of rice were spiked with parathion standard from 0ng/ml to5000ng/ml in double time, respectively, prepared simply. The results were obtained with the methods in 4 hours, and the recovery is from 81% to 103%, the average recovery is 89.3%. 5 samples of rice were measure with HPGC in other laboratories, and the coincidence rate comparing of the methods and HPGC are from 76% to 100%, the average coincidence rate is 89.8%.(3) Precision test: 5 samples of rice were spiked with parathion standard from Ong/rnl to 5000ng/ml and were measured on 7 test lots, with statistical treatment, the between-assay average percent coefficient of variations is 6.1%. 5 samples of rice were measured by 3 people at the same laboratory, the within-laboratory average percent coefficient of variations is 5.2%.Above tests showed that the method is simple, rapid, sensitive and precise, it fit for determination of parathion residues in rice. The results of looking related date base showed that this method is set up at first time in China.更多还原